A panax notoginseng cyclic peptide extract and a preparation method thereof

Abstract

The present application relates to a kind of panax notoginseng cyclic peptide extract and preparation method thereof, belong to natural active ingredient extraction technical field.The preparation method of the panax notoginseng cyclic peptide extract includes the following steps: panax notoginseng is crushed to obtain panax notoginseng powder, panax notoginseng powder is soaked in water to obtain panax notoginseng infusion, composite inoculant is inoculated to panax notoginseng infusion and is fermented to obtain fermentation product, then panax notoginseng cyclic peptide extract is extracted, and panax notoginseng cyclic peptide extract is obtained after freeze-drying;The composite inoculant is the composite inoculant of bacillus subtilis and butyric acid clostridium, and the mass ratio of bacillus subtilis and butyric acid clostridium in the composite inoculant is 1:1~3.The present application extracts panax notoginseng cyclic peptide extract by the composite inoculant of bacillus subtilis and butyric acid clostridium fermentation culture, and is safe and environmentally friendly, avoids the use of organic solvent, and meets the concept of green environmental protection.

Description

Technical Field

[0001] This invention belongs to the field of natural active ingredient extraction technology, specifically relating to a cyclic peptide extract of Codonopsis pilosula and its preparation method. Background Technology

[0002] *Pseudostellaria heterophylla*, belonging to the Caryophyllaceae family, is a traditional Chinese medicine. It has a sweet and slightly bitter taste, is neutral in nature, and enters the spleen and lung meridians. It is used to invigorate qi and strengthen the spleen, promote fluid production and moisten the lungs, and is indicated for spleen deficiency with fatigue, loss of appetite, post-illness weakness, qi and yin deficiency, spontaneous sweating with thirst, and dry cough due to lung dryness. Current research indicates that *Pseudostellaria heterophylla* contains polysaccharides, cyclic peptides, saponins, and other components, possessing anti-fatigue, anti-hypoxia, anti-wrinkle, and anti-aging effects. Cyclic peptides are its characteristic components. To promote the development of the *Pseudostellaria heterophylla* industry and develop related functional products and skincare products containing cyclic peptides, effectively obtaining extracts containing these peptides has become a pressing issue. Current extraction methods for *Pseudostellaria heterophylla* cyclic peptides generally involve alcohol extraction and silica gel column chromatography, which are costly and use large amounts of organic solvents, thus not conforming to the principles of green environmental protection. Summary of the Invention

[0003] The first objective of this invention is to provide a method for preparing Codonopsis pilosula cyclic peptide extract to solve the technical problem of high production costs when extracting Codonopsis pilosula cyclic peptide extract by alcohol extraction and silica gel column chromatography.

[0004] The second objective of this invention is to provide a cyclic peptide extract of Codonopsis pilosula.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a cyclic peptide extract of Codonopsis pilosula includes the following steps: pulverizing Codonopsis pilosula to obtain Codonopsis pilosula powder; soaking the Codonopsis pilosula powder in water to obtain Codonopsis pilosula soaking solution; inoculating the Codonopsis pilosula soaking solution with a compound microbial agent for fermentation culture to obtain fermentation product; subsequently extracting the Codonopsis pilosula cyclic peptide extract; and freeze-drying to obtain the Codonopsis pilosula cyclic peptide extract; wherein the compound microbial agent is a compound microbial agent of Bacillus subtilis and Clostridium butyricum, and the mass ratio of Bacillus subtilis to Clostridium butyricum in the compound microbial agent is 1:1 to 3.

[0007] Furthermore, the inoculation amount of the compound microbial agent is 10-15%.

[0008] Furthermore, the fermentation culture temperature is 33–37 °C, the fermentation culture time is 30–50 h, and the fermentation culture pH is 6–8.

[0009] Furthermore, the extraction method is as follows: extract at 30-40 °C on a constant temperature shaker at 200-300 rpm for 0.5-1 h, followed by centrifugation at 5000-6000 r / min for 10-30 min.

[0010] Furthermore, before extraction, the concentration of the fermentation product is diluted to 30–50 g / L.

[0011] Furthermore, the mass-to-volume ratio of the Codonopsis pilosula powder to water is 1 g: 10-15 mL, and the soaking time is 30-90 min.

[0012] Furthermore, the particle size of the Codonopsis pilosula powder is 20-30 mesh.

[0013] Furthermore, the freeze-drying method is as follows: pre-freezing at atmospheric pressure and -30 to -50 °C for 30 to 60 min, followed by drying under vacuum and -70 to -80 °C for 30 to 60 min.

[0014] A cyclic peptide extract of Codonopsis pilosula was prepared using the above-mentioned method for preparing cyclic peptide extracts of Codonopsis pilosula.

[0015] The beneficial effects of this invention are:

[0016] This invention extracts cyclic peptides from Codonopsis pilosula through fermentation culture using a compound bacterial agent of Bacillus subtilis and Clostridium butyricum. This method is safe and environmentally friendly, avoids the use of organic solvents, and conforms to the concept of green environmental protection.

[0017] This invention utilizes a combination of Bacillus subtilis and Clostridium butyricum to achieve a high extraction rate of Codonopsis pilosula cyclic peptide extracts. Furthermore, the extracted Codonopsis pilosula cyclic peptide extracts show a significant increase in the content of Codonopsis pilosula cyclic peptide A and Codonopsis pilosula cyclic peptide B. The Codonopsis pilosula cyclic peptide extracts prepared by this invention can be used in anti-wrinkle skincare products.

[0018] As can be seen from Comparative Examples 1 and 2 of the present invention, the extraction rate of Codonopsis pilosula cyclic peptide extract obtained by single-strain fermentation is low, and the content of Codonopsis pilosula cyclic peptide A and Codonopsis pilosula cyclic peptide B in the extracted Codonopsis pilosula cyclic peptide extract is also low. As can be seen from Comparative Example 4 of the present invention, the extraction rate and the content of Codonopsis pilosula cyclic peptide extract obtained by fermentation using a compound bacterial agent of Bacillus subtilis and Lactobacillus are also low. Using a compound bacterial agent of any two bacteria can not always achieve the effect of the present invention. In the present invention, Bacillus subtilis and Clostridium butyricum have a compounding effect. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments thereof.

[0020] The preservation number of Clostridium butyricum is CCTCC NO: M2024807.

[0021] The preservation number of Bacillus subtilis is CGMCC No. 29125.

[0022] The preservation number of Lactobacillus is CGMCC NO.23158. Example 1

[0023] The preparation method of the Codonopsis pilosula cyclic peptide extract in Example 1 is as follows:

[0024] 1 g of *Codonopsis pilosula* was pulverized to obtain *Codonopsis pilosula* powder with a mesh size of 20 mesh. 1 g of *Codonopsis pilosula* powder was soaked in 10 mL of water for 30 min to obtain a *Codonopsis pilosula* soaking solution. The soaking solution was sterilized at 121 ℃. Then, a compound bacterial agent was inoculated into the soaking solution at an inoculation rate of 12% (v / v). Fermentation was carried out in a fermentation chamber at 35 ℃ for 50 h at a pH of 7. After fermentation, the fermentation product was inactivated. The concentration of the fermentation product was adjusted to 30 g / L with ultrapure water. Extraction was carried out on a constant temperature shaker at 30 ℃ and 200 rpm for 0.5 h. Then, the product was centrifuged at 5000 r / min for 15 min, and the supernatant was collected as the *Codonopsis pilosula* cyclic peptide extract. The *Codonopsis pilosula* cyclic peptide extract was pre-frozen at -30 ℃ under normal pressure for 30 min, and then dried under vacuum at -70 ℃ for 60 min to obtain the *Codonopsis pilosula* cyclic peptide extract. The mass ratio of Bacillus subtilis to Clostridium butyricum in the compound microbial agent is 1:2. The *Pseudostellaria heterophylla* used in Example 1 originated from Guizhou Province. Example 2

[0025] The preparation method of the Codonopsis pilosula cyclic peptide extract in Example 2 is as follows:

[0026] 3 g of *Codonopsis pilosula* was pulverized to obtain *Codonopsis pilosula* powder with a mesh size of 20 mesh. The powder was soaked in 45 mL of water for 90 min to obtain a *Codonopsis pilosula* soaking solution. The soaking solution was sterilized at 121 ℃. Then, a compound bacterial agent was inoculated into the soaking solution at an inoculation rate of 15% (v / v). Fermentation was carried out in a fermentation chamber at 37 ℃ for 45 h at a pH of 8. After fermentation, the fermentation product was inactivated. The concentration of the fermentation product was adjusted to 50 g / L with ultrapure water. Extraction was carried out on a constant temperature shaker at 40 ℃ and 200 rpm for 0.5 h. Then, the product was centrifuged at 6000 r / min for 15 min, and the supernatant was collected as the *Codonopsis pilosula* cyclic peptide extract. This extract was pre-frozen at -50 ℃ under normal pressure for 30 min, and then dried under vacuum at -80 ℃ for 30 min to obtain the *Codonopsis pilosula* cyclic peptide extract. The mass ratio of Bacillus subtilis and Clostridium butyricum in the compound microbial agent was 1:1. The *Pseudostellaria heterophylla* used in Example 2 originated from Guizhou Province. Example 3

[0027] The preparation method of the Codonopsis pilosula cyclic peptide extract in Example 3 is as follows:

[0028] 100 g of *Pseudostellaria heterophylla* was pulverized to obtain *Pseudostellaria heterophylla* powder with a mesh size of 30 mesh. The powder was soaked in 1000 mL of water for 60 min to obtain a *Pseudostellaria heterophylla* soaking solution. This soaking solution was sterilized at 121 ℃. Subsequently, a compound bacterial agent was inoculated into the soaking solution at an inoculation rate of 10% (v / v). Fermentation was carried out in a fermentation chamber at 33 ℃ for 50 h at a pH of 7. After fermentation, the fermentation product was inactivated. The concentration of the fermentation product was adjusted to 40 g / L with ultrapure water. Extraction was performed on a constant-temperature shaker at 30 ℃ and 300 rpm for 0.5 h. The product was then centrifuged at 6000 r / min for 30 min, and the supernatant was collected as the *Pseudostellaria heterophylla* cyclic peptide extract. This extract was pre-frozen at -40 ℃ under normal pressure for 60 min, and then dried under vacuum at -80 ℃ for 60 min. min, which refers to the cyclic peptide extract of Codonopsis pilosula. The mass ratio of Bacillus subtilis and Clostridium butyricum in the compound bacterial agent is 1:3. The Codonopsis pilosula in Example 3 is from Guizhou.

[0029] Examples 4-6

[0030] The preparation methods of the Codonopsis pilosula cyclic peptide extracts in Examples 4-6 are roughly the same as those in Examples 1-3. The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Example 4 and that in Example 1 is that the Codonopsis pilosula in Example 4 is produced in Nanjing.

[0031] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Example 5 and that in Example 2 is that the Codonopsis pilosula in Example 5 is produced in Nanjing.

[0032] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Example 6 and that in Example 3 is that the Codonopsis pilosula in Example 6 was produced in Nanjing.

[0033] Comparative Example 1

[0034] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 1 and Example 1 is that the compound bacterial agent in Comparative Example 1 is replaced with Bacillus subtilis, and the inoculation of Bacillus subtilis is 15% (v / v).

[0035] Comparative Example 2

[0036] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 2 and Example 1 is that the compound bacterial agent in Comparative Example 2 is replaced with Clostridium butyricum, and the inoculation of Clostridium butyricum is 15% (v / v).

[0037] Comparative Example 3

[0038] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 3 and Example 1 is that the mass ratio of Bacillus subtilis and Clostridium butyricum in Comparative Example 3 is 1:4.

[0039] Comparative Example 4

[0040] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 4 and Example 1 is that the compound bacterial agent in Comparative Example 4 is Bacillus subtilis and Lactobacillus.

[0041] Comparative Examples 5-8

[0042] The preparation methods of the cyclic peptide extracts of Codonopsis pilosula in Comparative Examples 5-8 are roughly the same as those of the cyclic peptide extracts of Codonopsis pilosula in Comparative Examples 1-4. The difference between the preparation method of the cyclic peptide extract of Codonopsis pilosula in Comparative Example 5 and that in Comparative Example 1 is that the Codonopsis pilosula in Comparative Example 5 is produced in Nanjing.

[0043] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 6 and that in Comparative Example 2 is that the Codonopsis pilosula in Comparative Example 6 was produced in Nanjing.

[0044] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 7 and that in Comparative Example 3 is that the Codonopsis pilosula in Comparative Example 7 was produced in Nanjing.

[0045] The difference between the preparation method of the Codonopsis pilosula cyclic peptide extract in Comparative Example 8 and that in Comparative Example 4 is that the Codonopsis pilosula in Comparative Example 8 was produced in Nanjing.

[0046] The extraction rates of the cyclic peptide extracts of Codonopsis pilosula in Examples 1-6 and Comparative Examples 1-8 are shown in Table 1.

[0047] Table 1. Extraction rates of Codonopsis pilosula cyclic peptide extracts from Examples 1-6 and Comparative Examples 1-8

[0048] Sample Extraction rate of the Pseudostellaria heterophylla cyclic peptide extract (%) Example 1 95.2 Example 2 96.7 Example 3 94.9 Example 4 98.2 Example 5 97.6 Example 6 95.8 Comparative Example 1 90.3 Comparative Example 2 91.1 Comparative Example 3 88.7 Comparative Example 4 76.5 Comparative Example 5 87.7 Comparative Example 6 80.3 Comparative Example 7 86.9 Comparative Example 8 80.2

[0049] As shown in Table 1, the extraction rate of *Codonopsis pilosula* cyclic peptide extract using a compound inoculum of *Bacillus subtilis* and *Clostridium butyricum* was significantly higher than that using a single inoculum. Furthermore, the extraction rate decreased when the mass ratio of *Bacillus subtilis* to *Clostridium butyricum* was 1:4. Moreover, the extraction rate was even lower when the compound inoculum was replaced with a compound inoculum of *Bacillus subtilis* and *Lactobacillus*. Replacing *Clostridium butyricum* with *Lactobacillus* did not result in any synergistic effect between *Bacillus subtilis* and *Lactobacillus*. Compared to single-strain fermentation, the extraction rate of *Codonopsis pilosula* cyclic peptide extract obtained using fermentation with *Bacillus subtilis* and *Lactobacillus* was lower, indicating a synergistic effect between *Bacillus subtilis* and *Clostridium butyricum*.

[0050] The contents of ginsenoside A and ginsenoside B in the cyclic peptides of *Codonopsis pilosula* were determined using a Hewlett Packard HP Series 1100 high-performance liquid chromatograph. The contents of ginsenoside A and ginsenoside B in Examples 1-3 and Comparative Examples 1-4 are shown in Table 2, and the contents of ginsenoside A and ginsenoside B in Examples 4-6 and Comparative Examples 5-8 are shown in Table 3.

[0051] Table 2. Results of determination of the content of Pseudostellaria heterophylla cyclic peptide A and Pseudostellaria heterophylla cyclic peptide B in Examples 1-3 and Comparative Examples 1-4.

[0052] Sample Pseudostellaria heterophylla cyclic peptide A (μg / g) Pseudostellaria heterophylla cyclic peptide B (μg / g) Example 1 206.1 292.7 Example 2 239.2 287.5 Example 3 235.9 296.1 Comparative Example 1 104.3 180.3 Comparative Example 2 187.6 187.9 Comparative Example 3 155.8 163.5 Comparative Example 4 173.7 209.1

[0053] As shown in Table 2, the contents of ginseng cyclic peptides A and B in the ginseng cyclic peptide extract obtained by fermentation using the compound bacterial agent of Bacillus subtilis and Clostridium butyricum of the present invention are significantly increased compared with the ginseng cyclic peptide extract obtained by single bacterial fermentation. At the same time, compared with the ginseng cyclic peptide extract obtained by fermentation using the compound bacterial agent of Bacillus subtilis and Lactobacillus in Comparative Example 4, the contents of ginseng cyclic peptide A and B in the cyclic peptide extract obtained by fermentation using the compound bacterial agent of Bacillus subtilis and Clostridium butyricum are also significantly increased, indicating that Bacillus subtilis and Clostridium butyricum have a compound effect in the present invention.

[0054] Table 3. Results of determination of the content of Pseudostellaria heterophylla cyclic peptide A and Pseudostellaria heterophylla cyclic peptide B in Examples 4-6 and Comparative Examples 5-8.

[0055] Sample Pseudostellaria heterophylla cyclic peptide A (μg / g) Pseudostellaria heterophylla cyclic peptide B (μg / g) Example 4 135.1 98.1 Example 5 129.6 105.8 Example 6 129.4 112.5 Comparative Example 5 67.4 76.3 Comparative Example 6 89.3 82.0 Comparative Example 7 65.2 70.3 Comparative Example 8 79.3 77.4

[0056] As can be seen from Tables 2 and 3, the preparation method of the Codonopsis pilosula cyclic peptide extract of the present invention is applicable to Codonopsis pilosula from different sources, and can increase the content of Codonopsis pilosula cyclic peptide A and Codonopsis pilosula cyclic peptide B in Codonopsis pilosula. The preparation method of the Codonopsis pilosula cyclic peptide extract of the present invention is applicable to Codonopsis pilosula from different sources.

[0057] Application Example 1

[0058] An anti-wrinkle skincare product includes the following ingredients:

[0059] In Example 1, 2.5 kg of Codonopsis pilosula cyclic peptide extract, 1 kg of glycerin, 0.5 kg of allantoin, 3 kg of sodium hyaluronate, 1.5 kg of squalane, 2 kg of isononyl isononanoate, 0.8 kg of cetearyl alcohol, 0.5 kg of polyglycerol-10 stearate, 1.5 kg of xanthan gum, and 0.01 kg of p-hydroxyacetophenone were added to deionized water to make up to 100 kg.

[0060] The preparation method of the anti-wrinkle skincare product in Application Example 1 is as follows:

[0061] The formula amounts of glycerin, allantoin, sodium hyaluronate, xanthan gum, and water were stirred evenly at 50 ℃ and 300 rpm to obtain phase A. The formula amounts of squalane, isononyl isononanoate, cetearyl alcohol, and polyglycerol-10 stearate were stirred evenly at 50 ℃ and 300 rpm to obtain phase B. Phase B was added to phase A, emulsified and homogenized, cooled to 25 ℃, and the Codonopsis pilosula cyclic peptide extract and p-hydroxyacetophenone from Example 1 were added and stirred evenly to obtain the final product.

[0062] Application Examples 2-9

[0063] The preparation methods of the skin care products in Application Examples 2-9 are roughly the same as those in Application Example 1. The difference between the skin care products in Application Examples 2-9 and Application Example 1 is that the Codonopsis pilosula cyclic peptide extract in Application Examples 2-9 is replaced with the Codonopsis pilosula cyclic peptide extract in Comparative Examples 1-8.

[0064] Test Example 1

[0065] 1. Test substance: Anti-wrinkle skin care products used in Examples 1 to 9; Blank: No substance added to the blank group.

[0066] 2. Basis: 2015 edition of "Cosmetic Safety Technical Specifications", GB 17149.1—1997 "Diagnostic Criteria and Treatment Principles for Cosmetic Skin Diseases", and GB 17149.2—1997 "Diagnostic Criteria and Treatment Principles for Cosmetic Contact Dermatitis".

[0067] 3. Methods: 270 volunteers were selected and divided into 9 groups of 30 each. The flexor surface of the volunteers' forearms was used as the test site. The test substance and a blank were applied to the skin using the methods described in the 2015 edition of the "Cosmetic Safety Technical Specifications" for 24 hours. Skin reactions were observed at 30 minutes, 24 hours, and 48 hours after the test substance was removed, and the results were recorded.

[0068] 4. Test results: The results of the human skin closed patch test showed that among the 270 people who used the anti-wrinkle skin care products prepared in Examples 1 to 9, 0 cases showed positive reactions. The test products did not cause adverse reactions on the skin of this batch of volunteers.

[0069] Test Example 2

[0070] After cleansing their faces morning and evening, volunteers applied 2 g of the anti-wrinkle skincare product to their faces and massaged for 2-3 minutes. All volunteers underwent wrinkle-reducing effect testing on days 0, 30, and 60 after cleansing. The wrinkle-reducing effect test used a VISIA-CR facial image analyzer to capture skin images and measure the proportion of wrinkles. The wrinkle proportion difference refers to the difference between the wrinkle proportion after using the anti-wrinkle skincare product and the wrinkle proportion before using the anti-wrinkle skincare product.

[0071] Table 4. Results of the wrinkle removal test

[0072]

[0073] As can be seen from Table 4, the volunteers who used the anti-wrinkle skin care product of Application Example 1 of the present invention had a high rate of wrinkle reduction, indicating that the content of Codonopsis pilosula cyclic peptide A and Codonopsis pilosula cyclic peptide B in the Codonopsis pilosula cyclic peptide extract increased after fermentation with the compound microbial agent of the present invention, thereby achieving a good anti-wrinkle effect.

Claims

1. A preparation method of a Pseudostellaria heterophylla cyclic peptide extract, characterized in that, The method comprises the following steps: The step of obtaining the panax notoginseng powder by crushing the panax notoginseng, the step of obtaining the panax notoginseng soaking liquid by soaking the panax notoginseng powder in water, the step of obtaining the fermentation product by inoculating the composite microbial agent into the panax notoginseng soaking liquid and then culturing, the step of obtaining the panax notoginseng cyclic peptide extract by extracting the fermentation product with ultrapure water, and the step of obtaining the panax notoginseng cyclic peptide extract by freeze-drying; the composite microbial agent is a composite microbial agent of bacillus subtilis and clostridium butyricum, the mass ratio of bacillus subtilis to clostridium butyricum in the composite microbial agent is 1:1-3, the preservation number of clostridium butyricum is CCTCC NO: M2024807, and the preservation number of bacillus subtilis is CGMCC No. 29125.

2. The method of claim 1, wherein the PCTs are extracted from P. notoginseng. The inoculation amount of the composite microbial agent is 10-15%.

3. The method of claim 1, wherein the extraction is performed by using water, ethanol, methanol, or a mixture thereof. The fermentation culture temperature is 33-37 DEG C, the fermentation culture time is 30-50 h, and the fermentation culture pH is 6-8.

4. The method of claim 1, wherein the step of extracting the PPTs is performed by using a solvent selected from the group consisting of water, methanol, ethanol, and a mixture thereof. The extraction method is as follows: the extraction is performed at 30-40 DEG C by using a constant-temperature shaking table at a rotation speed of 200-300 rpm for 0.5-1 h, and then centrifugation is performed at 5000-6000 r / min for 10-30 min.

5. The method of claim 1 or 4, wherein the extraction is performed by using water, ethanol, methanol, acetone, acetic acid, or a mixture thereof. Before extraction, the concentration of the fermentation product is diluted to 30-50 g / L.

6. The method of claim 1, wherein the extraction is performed by using water, ethanol, methanol, or a mixture thereof. The mass-volume ratio of the panax notoginseng powder to water is 1 g: 10-15 mL, and the soaking time is 30-90 min.

7. The method of claim 1, wherein the extraction is performed by using water, ethanol, methanol, or a mixture thereof. The mesh number of the panax notoginseng powder is 20-30.

8. The method of claim 1, wherein the extraction is performed by using water, ethanol, methanol, or a mixture thereof. The freeze-drying method is as follows: pre-freezing at normal pressure and at-30--50 DEG C for 30-60 min, and then drying at vacuum and at-70--80 DEG C for 30-60 min.

9. A Pseudostellaria heterophylla cyclic peptide extract, characterized in that, The panax notoginseng cyclic peptide extract is prepared by using the preparation method of claim 1.

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